Orange juice containing Pediococcus acidilactici CE51 modulates the intestinal microbiota and reduces induced inflammation in a murine model of colitis

The management of inflammatory bowel diseases has been widely investigated, especially ulcerative colitis. Thus, studies with the application of new probiotic products are needed in the prevention/treatment of these clinical conditions. The objective of this work was to evaluate the effects of probiotic orange juice containing Pediococcus acidilactici CE51 in a murine model of colitis. 45 male Swiss lineage mice were used, divided into five groups (n = 9): control, colitis, colitis + probiotic (probiotic orange juice containing CE51), colitis + placebo (orange juice) and colitis + sulfasalazine (10 mg/kg/Weight). The induction of colitis was performed with dextran sodium sulfate (3%). The treatment time was 5 and 15 days after induction. Histopathological analysis, serum measurements of TNF-α and C-reactive protein and metagenomic analysis of feces were performed after euthanasia. Probiotic treatment reduced inflammation in the small intestine, large intestine and spleen. The probiotic did not alter the serum dosages of TNF-α and C-reactive protein. Their use maintained the quantitative ratio of the phylum Firmicutes/Bacteroidetes and increased Lactobacillus helveticus with 15 days of treatment (p < 0.05). The probiotic orange juice containing P. acidilactici CE51 positively modulated the gut microbiota composition and attenuated the inflammation induced in colitis.


Animals and experimental groups
Male Mus musculus (swiss) eight-week-old mice weighing 34.5 to 54.0 g, from the animal house of the University of Western Sao Paulo were used.The animals were kept in polypropylene boxes, on a shelf with an air filtration system and temperature and relative humidity control (23 °C; 60-70% humidity), light (12 h light/dark cycle), fed with mice feed (NUVILAB CR-1, QUIMTIA, Brazil) and filtered drinking water ad libitum 19,20 and acclimated to the conditions for at least seven days before experimental manipulation.The experiments took place during the clear phase of the cycle and were carried out in accordance with the approval of the Ethics Committee on the Use of Animals of University of Western Sao Paulo (CEUA/Unoeste-nº 5897).This study was carried out in compliance with the ARRIVE guidelines.A total of 45 mice were used, randomly distributed into five groups containing nine animals, divided as shown in Table 1.
The experimental colitis was chemically induced, through the administration of dextran sodium sulfate (DSS) (Sigma-Aldrich, St. Louis, MO, USA).The DSS (3%) was dissolved (15 g) in the filtered water (500 mL) supplied daily to the animals, and administered over a period of 6 days 21,22 .The animals were monitored daily for weight and clinical signs.
The severity of colitis was measured every day taking into account the three parameters and their respective scores of the disease activity index (DAI).The weight of the animals was evaluated by an analytical balance (Gehaka, BT8000, Brazil), classified by the scores: body weight loss (score 0, no weight loss compared to initial weight; (1) weight loss within 1-5%; (2) weight loss within 5-10%; (3) weight loss within 10-20%; (4) greater than 20% weight loss).The stool consistency by observation of the feces in the cages (score 0, normal-solid consistency); 2, loose stool but with some solidity; 4, diarrhea).For the analysis of occult blood in the feces, the Meyer Method was used in order to identify the presence of hemoglobin in the sample 23 .The presence of occult/ visible blood in the stools was also classified by the following score: score 0, negative; 2, fecal occult blood test positive; 4, gross bleeding 24,25 .
The animals in the probiotic (CLPR) and placebo (CLP) groups received 50 µL of the products under study daily, orally by means of a micropipette (Single channel Pipette, LABMATE Pro, Poland) (enough to guarantee a daily intake of 10 8 CFU of the probiotic microorganisms in probiotic orange juice group).The animals of the Sulfasalazine group (CLS) received the drug (Sigma-Aldrich, St. Louis, MO, USA) daily, orally through a micropipette (Single channel Pipette, LABMATE Pro, Poland) (10 mg/kg/weight).The Control (C) and Colitis (CL) groups did not receive any of the treatments, only water and food.After 5 days of treatment, four animals from each group were euthanized, and with 15 days, the remaining five animals from each group.For euthanasia, anesthetics Ketamine Hydrochloride (10%) (Rompun, Bayer HealthCare, Kansas, MO, USA and Xylazine Hydrochloride (2%) (Bayer, Leverkusen, Germany) were administered intraperitoneally (75 e 10 mg/kg).Laparotomy was performed and the organs of the gastrointestinal system (the small and large intestines, stomach, liver and spleen) were removed and opened longitudinally for later macroscopic and histological analysis.

Histological analysis
After each treatment period (5 and 15 days), the animals were euthanized.The small and large intestines were removed and then, the lengths were measured.The stomach, liver and spleen were also collected to evaluate the potential general effect of the induced inflammation in other parts of the body and possible toxic effect of probiotic juice.www.nature.com/scientificreports/All organs were cleaned (fats and tissues adhered to the wall were removed), washed in running water to remove feces, fixed by immersion in a solution of 10% buffered formalin for 48 h, processed routinely and embedded in paraffin.Histological sections of 5 μm thick were stained with Hematoxylin-eosin (HE) for analysis under light microscopy (NIKON Labophot, Japan) by one blind trained researcher to the treatment.
Histomorphometry analysis was performed, obtaining 3 selected sections of each animal.The height (μm) of 10 villi and 10 crypts of each animal was obtained by measuring the vertical distance from the upper extremity to the lower limit of each.The villus and crypt height values for each animal were represented by the mean values of the three histological sections 27 .
The images of the histological sections were captured in a 100 × magnification by a digital camera connected to a light microscope (LEICA DM750, Leica Microsystems, Germany).The digital images were analyzed using appropriate software (Image J® 1.50b, National Institutes of Health, USA) by one blind trained researcher 27 .

Serum dosage for TNF-α and C-reactive protein
The animals' blood was collected by cardiac puncture under anesthesia, and then centrifuged (15 min at 1000×g at room temperature) (FANEM®, 206 BL, Brazil).The serum was collected and stored at -20 °C until analysis.The concentration of TNF-α and C-reactive protein (CRP) were determined by the enzyme-linked immunosorbent assay (ELISA) with commercial kits (FineTest, Wuhan, China and Millipore, Billerica, MA, USA, respectively) with intra-and inter-assay variations (CV%) of < 8% and < 10% to TNF-α and ± 8% and ± 7% to CRP.Briefly, the serum samples were incubated with antibodies (anti-TNF or anti-PCR) and labelled with horseradish peroxidase for subsequent enzymatic reaction with tetramethylbenzidine.The reaction was stopped by the addition of acidic solution and the resulting absorbance was measured spectrophotometrically at 450 nm (SpectraMax® PLUS 384 Microplate Reader, MOLECULAR DEVICES, EUA).

Metagenomic analysis of the intestinal microbiota
A stool pool of the middle intestine of 4 (treatment = 5 days) and 5 (treatment = 15 days) mice per treatment (Control, Colitis, Probiotic, Placebo and Sulfasalazine) were sampled.The intestinal content (feces) of each animal was collected, stored individually in microtubes free of DNases and RNases and, immediately, frozen at -20 °C (Freezer Indrel Scientific, Brazil) for subsequent metagenomic analysis.DNA extraction and DNA sequencing analysis of the samples was performed by Neoprospecta Microbiome Technologies (Florianópolis, SC, Brazil) using a molecular technique based on the sequencing of the 16S rRNA gene, amplifying the V3-V4 regions using 341F/806R primers, through the Illumina MiSeq sequencer.Sequence of primers were 341F 5′-CCT AYG GGRBGCASCAG-3′ and 806R 5′-GGA CTA CNNGGT ATC TAAT-3′.The DNA concentration of the amplicon pool was estimated with Picogreen dsDNA assays (Invitrogen, USA), and then the pooled libraries were diluted for accurate quantification of qPCR using KAPA Library Quantification Kit for Illumina platforms (KAPA Biosystems, Woburn, MA, USA).
The library bank was adjusted to a final concentration of 11 pM and sequenced in a MiSeq system, using the Illumina primers provided in the kit.A 300 nt single-ended run was performed using a V2 × 300 sequencing kit.
Sequence data were processed and analyzed with QIIME [Quantitative Insights Into Microbial Ecology, version 2022.2.0 (https:// qiime2.org/)].On average, a total of 153,269 raw reads were sequenced.Initially, during the demultiplexing and trimming steps, low quality reads were removed, such as reads up to Q30, unsatisfactory length reads, and chimeras were removed with QIIME.After this process, the dataset contained an average of 106.918 raw reads.The clean readings were used in defining the Amplicon Sequence Variant (ASV).
To measure the rates present in the samples, a predictor model of the V3 and V4 region was used.Heatmaps and barplots of relative abundance of ASVs were generated with Python (version 3.7) through codes developed by the company ByMyCell Inova Simples Ltda.

Statistical analysis
All analyzed data were submitted to the normality test using the Kolmogorov-Smirnov test.The animal sample size calculation was performed according to Arifin and Zahiruddin 28 .
The comparison between two groups was performed using the Student's t test for unpaired data or the Mann-Whitney test.When three or more groups were compared, analysis of variance (ANOVA) was used, followed by the Newman-keuls post-test to verify the difference between the groups.The results were considered significant for p < 0.05.The statistical program used was GraphPad Prism 3.0.

Beneficial effect of probiotic orange juice on DAI analysis in DSS-induced colitis
Clinical signs, symptomatologic parameters and macroscopic damage were analyzed according to the disease activity index criteria.There was a change in stool consistency and the presence of visible and occult blood was observed for up to 5 days after induction of colitis with DSS.However, the parameters were more intense in the colitis, placebo and sulfasalazine groups.Despite the loss of two mice (1 colitis group and 1 placebo group), no significant difference was observed in the survival curve between the groups after 5 and 15 days of treatment (Fig. 2A,B).After 5 days of treatment, no significant changes were observed in the weight variation of experimental groups of animals during the period before induction of colitis and after treatment with probiotic, placebo and sulfasalazine (Fig. 2C).However, the colitis, probiotic, placebo groups showed a significant decrease in weight in comparison to control group after 15 days of treatment (Fig. 2D).

Attenuation of inflammation by probiotic orange juice treatment in histopathological analysis in DSS-induced colitis with 5 days of treatment
Histopathological analyzes were performed on the stomach, small intestine, large intestine, liver and spleen (Table 2 and Fig. 3).No significant changes were found for stomach and liver (data not shown).
The analysis of the small intestine evaluated parameters such as intensity of inflammation and villus measurements.With 5 days of treatment, a lower intensity of inflammation was observed in the probiotic and sulfasalazine group compared to the colitis group (p < 0.05).In the 15-day treatment, a 40% reduction in inflammation was observed in the sulfasalazine group and 20% in the probiotic group (p < 0.05).The measurements of intestinal villi showed a significant increase in the probiotic and placebo group after treatment for 5 days (p < 0.05).However, after 15 days of treatment, no significant changes were observed among the groups.
In the large intestine, the measurement of crypts, intensity of inflammation and presence of ulceration were evaluated.Crypt measurements were higher in the placebo group treated for 5 days (p < 0.05) and did not show significant differences with 15 days of treatment.There was a significant reduction of 25% in the intensity of inflammation and 25% of the presence of ulceration in the probiotic group compared to the colitis group after 5 days, however, with 15 days of treatment a more pronounced reduction was observed in the sulfasalazine group (p < 0.05).
White pulp hyperplasia was evaluated in the spleen.There was a 50% reduction in hyperplasia in the probiotic, placebo and sulfasalazine group after 5 days of treatment (p < 0.05).However, the reduction was 35, 50 and www.nature.com/scientificreports/55% respectively in the probiotic, placebo and sulfasalazine groups with treatment for 15 days compared to the untreated colitis group (p < 0.05).

Reduction of C-reactive protein levels after 15 days of treatment with P. acidilactici CE51 on DSS-induced colitis
The measurement of TNF-α demonstrated similarity between the groups during 5 and 15 days of treatment with probiotic, placebo or sulfasalazine (Fig. 4A).The treatment time impacted the levels of C-reactive protein, with a higher dosage being observed with a 5-day treatment for all groups that had colitis induction (p < 0.05).The sulfasalazine group with 15 days of treatment showed a significant reduction when compared with the other groups (p < 0.05) (Fig. 4B).

Probiotic orange juice positively modulates the gut microbial communities in DSS-induced colitis
Figure 5 shows the relationship between species diversity, the phylum Firmicutes/Bacteroidetes (F/B) ratio and the number of specimens in the Lactobacillaceae family.There was a significant increase in the diversity of bacterial species after 5 days of treatment for the probiotic group and colitis group (p < 0.05) (Fig. 5A).There were no significant changes after 15 days of treatment (Fig. 5B).
The quantitative ratio of the phylum Firmicutes/Bacteroidetes was reduced in the colitis, placebo and sulfasalazine group after 5 days of treatment (p < 0.05).After 15 days of treatment, a significant decrease was observed in all groups (p < 0.05).The number of specimens of the Lactobacillaceae family, represented by the size of the circle, was reduced in about 50% for the probiotic group after 5 days of treatment (p < 0.05).However, such a reduction was not observed after 15 days of treatment.
The relative quantity of bacterial species after 5 days of treatment had a predominance of Lactobacillus murinus in the control group and sulfasalazine with approximately 70% each (Fig. 5C).Colitis, probiotic and placebo groups had a significant reduction in Lactobacillus murinus and an increase in the proportion of Lactobacillus hominis and Lactobacillus reuteri.However, the probiotic group also showed an increase in Enterococcus faecalis (p < 0.05).
After 15 days of treatment, a reduction in the relative amount of Lactobacillus murinus was observed in the probiotic group compared to the control group (p < 0.05) (Fig. 5D).On the other hand, there was an increase in Lactobacillus hominis for the colitis, probiotic, placebo and sulfasalazine group (p < 0.05).In the probiotic group, a significant increase in Lactobacillus helveticus was also observed (p < 0.05).
Table 2. Histopathological analysis.The small intestine, large intestine and spleen were stained with Hematoxylin-eosin (HE) for analysis under light microscopy and the digital images were analyzed using appropriate software (Image J® 1.50b, National Institutes of Health, USA) by one blind trained researcher 19 .The experimental groups were divided into two different periods for treatment, with animals treated for 5 (n = 4) and 15 days (n = 5) with probiotic (orange juice containing Pediococcus acidilactici CE51), placebo (orange juice) or sulfasalazine (10 mg/kg/weight) after DSS administration (3%) to induce colitis.The control group was not subjected to the colitis protocol and treatment.The following parameters were also specifically analyzed to determine the level of inflammation resulting from inflammation caused by DSS, according to Pegoraro et al. 18 : Intestine (small and large): Interstitial inflammatory infiltrate (0 = absent, 1 = mild, 2 = moderate, 3 = intense); epithelial ulceration (0 = absent; 1 = present and small; 2 = present and extensive); Spleen: white pulp hyperplasia (0 = absent; 1 = present) and red pulp hyperplasia (0 = absent; 1 = present).

Discussion
In view of the need for studies on compounds that act in the treatment and prevention of ulcerative colitis, this study evaluated and found the positive effect of the probiotic drink containing P. acidilactici CE51 regarding the reduction of inflammatory parameters presented in histopathological analyzes and modulation of the gut microbiota composition.Adequate concentration and administration of probiotic strains is essential to induce their positive effect, requiring an inoculum of at least 10 6 CFU/mL of the live probiotic strain at the time of consumption 29 .In the present study, a concentration of 10 8 CFU was used/mL and the effectiveness of this administration in reducing the effects of induced colitis has been demonstrated.Similar results were obtained by Srutkova et al. 30 and Mansour et al. 14 , respectively for the use of 2 × 10 8 CFUs of B. longum ssp.Longum CCM 7952 (Bl 7952) and CCDM 372 (Bl 372) in the prevention of DSS-induced colitis, and orally daily dose containing CFU 10 9 Pediococcus acidilactici strains induction for colitis by acetic acid.
Histopathological analyzes showed that the group that received P. acidilactici CE51 for 5 days showed a decrease in the intensity of inflammation in the intestines, an increase in intestinal villi, absence of ulceration and a reduction in white pulp hyperplasia when compared to the untreated colitis group.After 15 days of treatment with the P. acidilactici CE51, the evidence was less pronounced.
The inflammatory process in the intestinal mucosa results in reduced villi, digestive, and absorptive activities.The probiotic strains compete with pathogenic microorganisms for binding sites, reducing the occurrence of diarrhea and favoring the absorption of nutrients, this mechanism directly influences the recovery of the intestinal mucosa, increasing the height of the villi 31 .This positive effect could be observed in the probiotic group treated for 5 days.
The white pulp present in the spleen is particularly lymphatic and plays a fundamental role in the immunological activity 32 , which justifies the hyperplasia of the white pulp, since this increase is directly related to the proliferation of leukocytes due to a severe inflammatory process induced in these animals.
Evidence suggests that UC leads to complications in gastric mucosal lesions, involving tissue inflammation and destruction, potentially causing gastritis and ulcers 33 .Kwon et al. 34 show that DSS-induced colitis is associated with adipose tissue dysfunction and disrupted hepatic lipid metabolism leading to hepatosteatosis and dyslipidemia in mice.However, in the present study there were no histopathological differences in both organs and tissues 35 .
Orange juice, alone, also had the potential to reduce inflammatory parameters in murine-induced colitis.There was a reduction in the intensity of inflammation in the small intestine, an increase in the height of the villi and crypts and a reduction in spleen hyperplasia.Citrus juice, especially orange juice, is associated with improved immunity, as it is a source of vitamin C, flavonoids and bioactive compounds, which together have a potential anti-inflammatory, antioxidant effect and improved endothelial function 36 .This fact highlights the importance of orange as a food matrix that can reduce inflammatory biomarkers and serve as an attractive strategy to counteract these types of stresses.
Although no evidence has been found in the literature using P. acidilactici CE51 in the treatment of ulcerative colitis, the results of improvement in the histological parameters, as well as aid in the protection of the epithelial barrier is in agreement to the findings of Duary et al. 37 .In that work, the anti-inflammatory and immunomodulatory efficacy of probiotic strains of Lactobacillus plantarum Lp91 was observed as a prevention of ulcerative colitis when compared to the untreated colitis group and the colitis group treated with L. plantarum CSCC5276.The animals received the live cells at 10 9 CFU resuspended in 25 μL in PBS by mouth 37 .
Wang et al. 38 verified the mixture (1:1, 10 9 CFU/mL) of Lactobacillus plantarum ZDY2013 and Bifidobacterium bifidum WBIN03 as an excellent strategy of antioxidant activity and immune stress relief in mice with DSS-induced colitis.Those animals were treated for 7 days with probiotics, with a reduction in pro-inflammatory cytokines, such as TNF-α, and an increase in antioxidant factors, such as SOD1, SOD2, GPX2.Groeger et al. 39 when using 1 × 10 10 CFU viable Bifidobacterium infantis 35624 in the treatment of gastrointestinal and nongastrointestinal inflammatory disorders, concluded that this strain reduced the levels of C-reactive protein and TNF-α, but had no impact on ulcerative colitis.
The fact that the measurement of TNF-α did not give statistical difference between the groups treated for 5 and 15 days in the present study, corroborates with a systematic review where it was found that the use of probiotics does not impact the measurements of serum IL10 and TNF-α 40 .Regarding the C-reactive protein dosage, a significant reduction was demonstrated only in the sulfasalazine group after 15 days of treatment.The dosages of C-reactive protein and TNF-α were not influenced by the administration of P. acidilactici CE51 probable because they are considered markers of systemic inflammation and due to the short term of administration of the CE51 probiotic 41 .Studies evaluating its application for a longer period of time or its concomitant use with sulfasalazine are necessary to further evaluate its potential to improve the imumodulatory response of colitis induced in animals.
Studies have shown that during an IBD there is a significant reduction in potentially beneficial species such as Bifidobacterium, Lactobacilli, and Firmicutes and Bacteroidetes phyla, which make up the healthy intestinal microbiota.On the other hand, there may be a large increase in pathogenic strains under IBD conditions such as: Campylobacter jejuni, Salmonella enterica, Vibrio cholera and Escherichia coli (E.coli) and Bacteroides fragilis 42 .
The gut microbiota is characterized as a diverse microbial community present in the gastrointestinal tract that maintains gut symbiosis in the host.In it, the two dominant bacterial phyla, Firmicutes and Bacteroidetes, are present, which consist of more than 90% of the total community.The relationship between the two is expressed as the Firmicutes/Bacteroidetes ratio, commonly related to intestinal pathological conditions 43,44  www.nature.com/scientificreports/ the microbiota is represented with relative absence of these phyla and with exacerbated presentation of Enterobacteriaceae and Fusobacteria 45 .
In intestinal biopsies and stool samples from UC, it is common to observe disproportions in the F/B ratio, in which Firmicutes are decreasing and Bacteroidetes are generally abundant 46 .In the present study, a significant decrease in F/B in the colitis, placebo and sulfasalazine groups treated for 5 days and maintaining the proportion in the probiotic group equivalent to the control group.This data highlights the maintenance of a healthy intestinal microbiota in the group that received P. acidilactici CE51, since the decrease in the abundance of Firmicutes and Bacteroidetes is related to the development and advancement of the disease, resulting from bacterial dysbiosis in inflamed sites compared to non-inflamed tissues 47 .
Strains of the Lactobacillaceae and Bifidobacteriaceae families are characterized as the most common probiotics.The family Lactobacillaceae (phylum Firmicutes) is predominant in the gut microbiota, comprising 31 genera, principally Lactobacillus species.The presence of Lactobacillaceae in the gut is important for preventing colonization by enteropathogenic bacteria through competitive adhesion and aggregation to binding sites.In addition, they demonstrate a protective effect on the intestinal tract, as they increase immunity and produce antimicrobial substances that inhibit pathogens, thus exerting antagonism against them 48,49 .
In the intestinal lumen, microbial genera predominate that can be identified in the feces, such as Bacteroides, Bifidobacterium, Streptococcus, Enterobacteriacae, Enterococcus, Clostridium, Lactobacillus and Ruminococcus 50 .The number of specimens from the Lactobacillaceae family showed a significant reduction after the 5-day period, possibly attributed to an increase in the species Enterococcus faecalis belonging to the Enterococcaceae family.Probably, this fact may be related to the condition of competition of different bacterial species for nutrients and adhesion to the mucosa in this condition 51 .
The results presented here did not show the presence of P. acidilactici CE51 in fecal samples, and this may be related to the application of the metagenomic technique, which has sensitivity limitations when it comes to a complex sample and which presents a microbiota with a high diversity of species with possible inhibitors; as is the case with stool analysis 52 .In addition, the bacteria may have been unable to colonize and compete with the murine gastrointestinal microbiota.Similar results were observed in the study by Grimm et al. 53 , where there was treatment for B. bifidum S17/pMGC (2 × 10 9 CFU in 20 μL of PBS), however, fecal levels were decreased during the pretreatment and DSS phase, making it difficult to detect them in the feces.
It was possible to observe a positive modulation of the species that make up the intestinal microbiota with the treatment of the P. acidilactici CE51.It is known that Lactobacillus murinus is the most prevalent and prevalent isolate found in this rodent species chosen for the experiment, as demonstrated in the control and sulfasalazine group with 5 days of treatment 54 .When analyzing the animals treated for 15 days, it was noted that all groups of animals showed an increase in Lactobacillus hominis, one of the most common species found in the intestine 55 .In addition, the group that received P. acidilactici CE51 also had a significant and predominant increase in Lactobacillus helveticus.These microorganisms assist in intestinal positive modulation, can improve the inflammatory response and are considered effective in the treatment of diarrhea associated with antibiotics and liver damage 56 .
In the literature, the use of anti-inflammatory drugs and probiotic mixtures is highlighted as the main means of combating UC.Palumbo et al. 57 showed that patients treated with mesalazine and a mixture of probiotics containing Lactobacillus salivarius, Lactobacillus acidophilus and Bifidobacterium bifidus strain BGN4 (Acronelle®, Bromatech srl, Milan, Italy) showed significant improvement in relation to those treated with mesalazine alone.This demonstrates that the combination of therapies generates positive effects on the patient's health and the probiotic mixtures help in the effective improvement of the clinical condition, which can potentiate anti-inflammatory therapy.A study emphasized the potential of capsules containing 2.5-25 × 10 9 viable bacteria (Mutaflor 100 mg; Ardeypharm GmbH, Herdecke, Germany)of Escherichia coli Nissle 1917 (EcN) probiotic strain to maintain remission in patients with ulcerative colitis and be equivalent to the standard treatment with mesalazine 58 .Other researchers, applying the same strain in experiments with mice, reported the benefits in relation to pathological and/or physiological parameters, and daily 10 8 CFU of live bacteria and 1.5-2 × 10 8 CFU (Mutaflor mite, Ardeypharm, Germany) EcN twice daily by oral were applied, respectively 59,60 .
As a result of these data, a comparison can be established with the findings of the present study, since P. acidilactici CE51 was shown to be more effective than the treatment with the sulfasalazine drug in some parameters such as increased villi in the small intestine, reduced ulceration and intensity of inflammation in the large intestine and maintenance of the proportion of the phylum F/B when used for 5 days in the treatment.
Thus, these data suggest that the benefits obtained by the probiotic drink containing P. acidilactici CE51 are more evident in 5 days of treatment.After this period, the organism is probably able to obtain an inflammatory response for the recovery of tissue damage generated by the induction of colitis.Furthermore, it is worth mentioning that the intestine has an accelerated cell multiplication and renewal process, which may also favor its resourcefulness in recovery 61 .

Conclusion
In conclusion, the results showed the potential beneficial effect of the application of probiotic orange juice containing Pediococcus acidilactici CE51 as a promising therapy in the treatment and management of colitis in murine, being able to maintain the integrity of the intestinal barrier, maintain body weight, assist in the decline of DAI and positively modulated the gut microbiota.

Table 1 .Figure 1 .
Figure 1.Experimental design.The experiment was carried out with 45 animals divided in 5 experimental groups Control (C); Colitis (CL); Colitis + Probiotic (CLPR; Colitis + Placebo (CLP); Colitis + Sulfasalazine (CLS) for 37 days divided in the following stages: Adaptation period of 7 days (Blue line); T0/d0: Initiation of administration of probiotic orange juice containing P. acidilactici CE51 for the CLPR group, or orange juice (placebo) for the CLP group, which lasted until the last day of the experiment (Green line); T1/d09: Beginning of the period of induction of ulcerative colitis by DSS, which lasted 6 consecutive days (Yellow line); T2/d15: The arrow indicates the withdrawal of the DSS and the beginning of the treatments that lasted 5 and 15 days.In this period also started treatment with sulfasalazine (Orange line); T3/d20: Exact time when the first euthanasia occurred (Euthanasia 1) after treatment with probiotic orange juice, placebo or sulfasalazine for 5 days; T4/d30: Exact time when the second euthanasia (Euthanasia 2) occurred after treatment with probiotic orange juice, placebo or sulfasalazine for 15 days.d Day (Above the colored lines); T Time (Below the colored lines); Black arrow Induction of ulcerative colitis by DSS.

Figure 2 .
Figure 2. Beneficial effect of probiotic orange juice on DAI analysis in DSS-induced colitis.Survival curve (A, B) and weight variation (C, D) of mice treated with 5 days (left side) (n = 4) and 15 days (right side) (n = 5) with probiotic (orange juice containing Pediococcus acidilactici CE51), placebo (orange juice) or sulfasalazine (10 mg/ kg/weight) after DSS administration (3%) for induced colitis.The control group was not submitted to the colitis protocol.Comparison was done between all groups of each treatment period.*Statistically significant difference in weight variation between control group and groups: colitis, placebo and probiotic.
a Statistically significant difference in relation control group (p < 0.05).b Statistically significant difference in relation colitis group (p < 0.05).c Statistically significant difference in relation probiotic group (p < 0.05).d Statistically significant difference in relation placebo group (p < 0.05).e Statistically significant difference in relation sulfasalazine group (p < 0.05).

Figure 4 .Figure 5 .
Figure 4. Reduction of C-reactive protein levels after 15 days of treatment with P. acidilactici CE51 on DSSinduced colitis.Serum concentrations of TNF-α (A) and C-reactive protein (B) from mice treated for 5 days (n = 4) and 15 days (n = 5) with probiotic (orange juice containing Pediococcus acidilactici CE51), placebo (orange juice) and sulfasalazine (10 mg/kg/weight) after DSS administration (3%) to induce colitis.The concentration of TNF-α and C-reactive protein (CRP) were determined by the enzyme-linked immunosorbent assay (ELISA) and, in the final of the test, the resulting absorbance was measured spectrophotometrically at 450 nm.The control group was not submitted to the colitis protocol.*Statistical difference in relation to the control group (p < 0.05).a Statistical difference in relation to the colitis, placebo and probiotic group (p < 0.05).
. As the phyla Firmicutes and Bacteroidetes predominate in healthy intestines and help produce epithelial metabolites.In IBD,